Oils produced by plants can be found in a wide variety of products including soaps, lubricants, and foods. Interestingly, different plant species synthesize various oil types. For example, coconut and palm plants produce oils that are abundant in fatty acids having medium chain lengths (10-12 carbon atoms). These oils are used in the manufacture of soaps, detergents and surfactants and represent a US market size greater than $350 million per year. Other plants, such as rape, produce oils abundant in long chain fatty acids (22 carbon atoms) and are used as lubricants and anti-slip agents. Additional applications of plant oils include their use in plasticizers, coatings, paints, varnishes and cosmetics (Volker et al., (1992) Science 257:72-74; Ohlrogge, (1994) Plant Physiol. 104:821-826). However, the predominant use of plant oils is in the production of food and food products.
The characteristics of oils are determined predominately by the number of carbon atoms comprising the fatty acid chain. Most oils derived from plants are composed of varying amounts of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) fatty acids. Palmitic and stearic acids are 16- and 18-carbon long saturated fatty acids, respectively. Conventionally, they are designated as "saturated" since the fatty acid chains have no double bonds and therefore contain the maximal number of hydrogen atoms possible. Saturated fatty acids are linear molecules and tend to form self-stacked structures thereby resulting in high melting temperatures. For example, animal fats, which are solid at room temperature, are typically high in saturated fatty acids. The other predominant fatty acids found in plant oils, oleic, linoleic, and linolenic, are 18-carbon long fatty acid chains having one, two, and three double bonds therein, respectively. Oleic acid is typically considered a mono-unsaturated fatty acid, whereas linoleic and linolenic are considered to be poly-unsaturated fatty acids. These fatty acid chains are nonlinear due to bending induced by the insertion of the double bond in the cis conformation. Double bond insertion decreases melting point due to the inability of the fatty acid molecules to self-stack. For example, vegetable oils, which are typically liquid at room temperature, are high in unsaturated fatty acids.
Over the years, vegetable oils have gradually replaced animal-derived oils and fats as the major source of dietary fat intake. However, saturated fat in most industrialized nations has remained at 15 to 20% of total caloric intake. The United States Department of Agriculture has recently recommended that saturated fats make up less than 10% of daily caloric intake. To facilitate consumer awareness, current labeling guidelines issued by the United States Food and Drug Administration now require total saturated fatty acid levels be less than 1.0 g per 14 g serving to receive the "low-sat" label and less than 0.5 g per 14 g serving to receive the "no-sat" label. This means that the saturated fatty acid content of plant oils would need be less than 7% and 1.75% to receive the "low sat" and "no sat" label, respectively. Therefore, there has been a surge in increased consumer demand for "low-sat" oils. To date, this has been met principally with canola oil, and to a much lesser degree with sunflower, and safflower oils.
The total saturated fatty acid level of corn oil, approximately 13.9%, does not meet the labeling guidelines discussed above. On average, corn oil is comprised of 11.5% palmitic acid, 2.2% stearic acid, 26.6% oleic acid, 58.7% linoleic acid, and 0.8% linolenic acid. Corn oil also contains 0.26 arachidic acid, a twenty-carbon saturated fatty acid (Dunlap et. al., (1995) J. Amer. Oil Chem. Soc. 72:981-987). The fatty acid composition of corn oil instills it with properties that are most desirable in edible oils. These include properties such as heat stability, flavor, and long shelf life. However, consumer demand for "low sat" oils has resulted in a significant decrease in corn oil utilization and thus market share. Therefore, a corn oil with low levels of saturated fatty acids is highly desirable in that it would meet the consumer demand for healthier oils while having most or all of the properties that made corn oil popular in the past and a preferred oil for many uses.
Although corn oil with low levels of saturated fatty acids is desirable, there is also a demand for corn oil having high levels of saturated fatty acids. For example, about half of the total consumption of vegetable oils is in the form of margarine and shortening. However, the use of corn oil for these products requires chemical modification of the oil due to its low melting point. Typically, an increased melting point is achieved through catalytic hydrogenation which increases the level of saturated fatty acids. In this process, hydrogen atoms are added at double bonds found in the fatty acid through the use of a catalyst. An additional side reaction that occurs during hydrogenation is the substantial conversion of the naturally occurring cis double bonds to the trans isomer, which is more stable. There have been some controversies regarding health risks associated with intake of oils containing trans double bonds. In a recent study, it was shown that a diet high in trans isomer consumption actually raised serum lipoprotein profiles and cholesterol levels (Mensink and Katan (1990) N. Eng. J. Med. 323:439-445). Therefore, production of oil containing a higher content of saturated fatty acids would reduce the need for hydrogenation in margarine and shortening production thereby reducing the content of trans isomers in the diet. In addition, partial hydrogenation typically increases cost an additional 2 to 3 cents per pound of oil. Therefore, a corn oil with naturally high saturates levels is also highly desirable for production of margarine and shortening since this would fulfill a market need while reducing manufacture cost.
Corn is typically not considered to be an oil crop as compared to soybean, canola, sunflower and the like. In fact, the oil produced by corn is considered to be a byproduct of the wet milling process used to extract starch. Because of this, there has been little interest in modifying the saturate levels of corn oil until that disclosed herein.
As disclosed herein, the saturate levels of fatty acids in corn oil can be altered by modifying the expression levels of .beta.-Ketoacyl-Acyl carrier protein Synthase II (KAS II). KAS II catalyzes the elongation of fatty acid intermediates from 16:0-acyl carrier protein (ACP) (palmitoyl-ACP) to 18:0-ACP (stearoyl-ACP) by the addition of a two carbon moiety from malonyl-ACP to 16:0-ACP. During fatty acid biosynthesis, KAS II competes with the palmitoyl-acyl carrier protein thioesterase (PTE) for 16:0-ACP substrate. PTE terminates chain elongation by hydrolyzing the ACP moiety from the fatty acid intermediate, thereby liberating free fatty acids which are ultimately incorporated into seed oil. In this way, PTE is in large part responsible for regulating the amount of 16:0 in the triacylglycerol fraction. However, the equilibrium established by the competition of PTE and KAS II for palmitoyl-ACP plays a large role in fatty acid profiles observed in corn oil.
Over-expression of KAS II in plants is a strategy to reduce the amount of 16:0 in seed oil by shifting the equilibrium of palmitoyl-ACP to stearoyl-ACP. An increase in KAS II concentration forces the carbon flux toward 18:0-ACP, which is rapidly converted to 18:1-ACP by stearoyl-ACP desaturase (delta-9 desaturase). The amount of 16:0-ACP available for the thioesterase is effectively depleted, and the amount of saturated fatty acid in seed oil in the form of 16:0 is reduced. Alternatively, down-regulation of KAS II in plants is a strategy to increase the amount of 16:0 in seed oil by shifting the equilibrium from stearoyl-ACP production to palmitoyl-ACP production.